The primary goal of this research is to identify gene products involved in intracellular cholesterol transport and regulation of cholesterol levels. Using isolated Chinese hamster ovary (CHO) cell mutants which are defective in LDL-trafficking we will identify factors involved in cholesterol movement and regulation. One mutant, 3-6, by complementation analysis was found to be in a separate class; and thus, designated ced2. Mutant 3-6 shows impaired LDL-stimulation of cholesterol esterification and LDL-suppression of HMG CoA reductase, but normal movement of LDL- cholesterol from the lysosomes to the plasma membrane. Specific aim 1 involves the identification of gene(s) that are defective in the mutant ced2/3-6 cell line. This aim will be achieved by isolating cDNAs that correct the defective phenotype in the mutant. Initially, mutant 3-6 cells will be transfected with a normal cDNA library in a mammalian expression vector. Cells with normal phenotype will be identified using a two-step screening process, and the cDNAs expressed in the cloned cell lines will be isolated using plasmid rescue techniques. Finally, sequencing the corresponding genes in normal and mutant CHO cells will allow identification of the gene defects causing mutant phenotypes. This work should reveal important information on gene products that play a direct role in cholesterol homeostasis. Specific aim 2 involves analysis of the biochemical phenotype of the ced2 complementation group. Analysis of cholesterol movement, esterification by ACAT, sensitivity to Amphotericin B, and cell growth will be done to determine how this gene defect alters key aspects of intracellular cholesterol transport and metabolism.